The industrial application of biotechnology is based on the more recent advances within the field of molecular biology and genetics. As is well known, one of the ways that genetic variability is maintained within a population is through recombination, a process involving the exchange of genetic information among different DNA molecules that results in a reshuffling of genes. To provide recombination in the field of genetic engineering, a vector is usually used. The most commonly used vector is the DNA plasmid, a small genetic element that permits microorganisms to store genetic information elsewhere than in the nucleus.
Thus, plasmids have become useful elements in many biotechnological applications these days. For example, to produce recombinant proteins, genetic engineering of cells is performed by introducing plasmids that carry a gene encoding a protein, which is not expressed in the native cell. Thereby, many proteins useful primarily in the medical and diagnostic fields are easily produced using methods that have become more or less routine methods.
Another use of plasmids as vectors is in the field of gene therapy, which is expected to be one of the fastest growing areas in the next decade. Gene therapy is a therapeutic strategy where nucleic acids are introduced into human cells in order to cure genetic defects e.g. cystic fibrosis. The first human gene therapy trials began in 1990, using an ex vivo strategy. In this approach, the patient cells are harvested and cultivated in the laboratory and then incubated with vectors, such as plasmids, to introduce the therapeutic genes. Even though alternative approaches for delivering genes based on in vivo gene therapy, wherein a viral vector is directly administered to the patient, have been suggested more recently, the plasmid is expected to retain its importance in gene therapy. Thus, the increased use of such applications results in a need for large quantities of plasmid DNA. To this end, an efficient large-scale purification process, which can meet specifications in purity and quantitation, is required.
Conventionally, the production of plasmid DNA involves fermentation, primary purification and high-resolution separation.
Thus, firstly, the fermentation step commonly comprises to produce the plasmid DNA in bacteria, such as Escherichia coli, and will also involve a step for release of plasmid DNA from the bacterial cells known as lysis. In general, cell lysis can be achieved by a variety of chemical or mechanical methods, such as by addition of alkali or using a French press, respectively. However, for reasons of safety and to not harm the product, the alkaline lysis will be preferred in the production of plasmid DNA. Usually, several contaminants such as RNA, genomic DNA, proteins, cells and cells debris are released in such an alkaline lysis step.
Secondly, with regard to the primary purification step, methods such as two-phase systems, e.g. using polyethylene glycol (PEG) and a salt; temperature-induced phase separation using a thermoseparating polymer that separates into two phases at a to certain temperature; or size exclusion chromatography, sometimes denoted gel filtration, are commonly used.
With regard to the high-resolution separation step, chromatography is a commonly used technique. As is well known, the term chromatography embraces a family of closely related separation methods, which are all based on the principle that two mutually immiscible phases are brought into contact. More specifically, the target compound is introduced into a mobile phase, which is contacted with a stationary phase. The target compound will then undergo a series of interactions between the stationary and mobile phases as it is being carried through the system by the mobile phase. The interactions exploit differences in the physical or chemical properties of the components in the sample. The basis for the chromatographic principle known as ion exchange process is the competitive binding of ions of one kind, such as proteins or nucleic acids, for ions of another kind, such as salt ions, to an oppositely charged matrix known as the ion exchanger. The interaction between the target compound and the ion exchanger depends on several factors, such as net charge and surface charge distribution of the target compound, the ionic strength and the nature of the particular ions in the solvent, the proton activity (pH) etc.
Anion exchange chromatography has been suggested for purification of nucleic acids and plasmids. For example, WO 99/63076 (The Immune Response Corp.) discloses large scale plasmid purification using a single, “mixed mode” anion exchange step. The method disclosed requires a stringent ethanol wash to remove endotoxins and other impurities. More specifically, by increasing the amount of organic solvent in the wash step, the disclosed method shifts from a purely ionic mode to that of a “mixed” mode. The separation matrix used is e.g. triethylaminoethyl (TMAE) fractogel anion exchange resin (E.M. Science Fractogel TMAE Resin).
U.S. Pat. No. 6,270,970 (Smith et al) relates to mixed-bed solid phases for isolation of target nucleic acids, which are comprised of at least two different solid phases. Both phases bind the target nucleic acids, but under different solution conditions, and they release the nucleic acid under similar elution conditions. The solid phase of the different beds preferably comprise magnetic silica particles, and at least one preferably has an ion-exchange residue capable of exchanging with the target nucleic acid covalently attached to the surface of the support material. The term “surface” is stated to refer to the portion of the support material of a solid phase which comes into direct contact with a solution when the solid phase is combined therewith. Thus, the anion exchange ligands are present on external surfaces as well as on pore surfaces, as is evidenced by the statement that suitable anion-exchanger solid phases for use in the mixed-bed solid phases according to U.S. Pat. No. 6,270,970 are commercially available, illustrated e.g. by Sepharose™. The pore size of such commercial solid phases is commonly in a range that allows plasmids and similar size molecules to enter their interior.
Further, U.S. Pat. No. 6,441,160 (Tosoh Corp.) discloses plasmid purification using hydrophobic interaction chromatography (HIC), optionally combined with an anion exchange step. In the HIC step, protein and RNA are adsorbed at a salt concentration at which plasmids are not adsorbed to produce an eluate comprising plasmid and DNA. Thus, the liquid applied to the anion exchange column should not contain any RNA. A general disadvantage of using HIC is the requirement of high salt concentrations at the start, which due to crystallisation and precipitation are relatively difficult to handle. Suitable anion exchange separation matrices are stated to have a particle diameter of 2-500 μm and an average pore diameter of 1500-4000 Å. An illustrative anion exchange separation matrix is DEAE 5PW (Tosoh). However, U.S. Pat. No. 6,441,160 teaches that plasmid purification from a cleared lysate by means of anion exchange interaction chromatography alone is not advantageous, as evidenced by comparative Example 1 wherein it is concluded that many impurities were contained in the plasmid fraction obtained from a single anion exchange step. In said comparative example, a chromatography column having an inner diameter of 7.5 mm and a length of 7.5 cm was used.
U.S. Pat. No. 6,011,148 (Megabios Corp.) discloses a method for purification of nucleic acids, such as plasmid DNA, by circulating a plasmid containing solution through an ultrafiltration unit under conditions sufficient to allow a gel layer to form and filtering the solution through the ultrafiltration unit to provide a permeate solution and a retentate solution, whereby the nucleic acid is retained in the retentate solution. The filtration device used should have open channels, to avoid shear and decrease of yield of retained nucleic acid. An advantage of the method is that it avoids use of toxic chemicals and organic solvents, such as phenol, chloroform, ether etc, which may cause safety and regulatory concerns. Another advantage of the method is the high purity of the product obtained. The method can optionally be combined with a step of anion exchange for further purification, particularly from contaminating endotoxin, trace proteins, and residual cellular contaminants.
U.S. Pat. No. 6,214,586 (Genzyme Corp.) discloses a method for purifying plasmid DNA from a mixture containing plasmid DNA and genomic DNA comprising to treat a solution containing both plasmid DNA and genomic DNA with at least 80% by weight saturation with ammonium sulphate, thereby precipitating the genomic DNA and providing purified plasmid DNA in solution. The method may be combined with a step of reverse phase and anion exchange chromatography, in which case a preferred resin is Poros 50 DE2, a column of which is equilibrated preferably with a solution of 50 mM acetate, pH 5.4, 1 mM EDTA, 0.5 M NaCl, and 9.5% ethanol.
U.S. Pat. No. 6,313,285 (Genentech Inc.) discloses a process for purifying plasmid DNA from prokaryotic cells, wherein there is no use of enzymes to digest RNA. More specifically, the process comprises the steps of: (a) digesting the cells; (b) incubating the cells in the presence of alkali and a detergent to effect lysis and solubilisation thereof; (c) removing lysate contaminants to provide a plasmid DNA solution; (d) filtering the solution through a tangential flow filtration device to obtain a retentate containing the plasmid DNA; and (e) collecting the retentate. The process may comprise a subsequent step of anion exchange chromatography.
U.S. Pat. No. 6,242,220 (Qiagen GmbH) discloses an improved protocol for separation of ccc DNA from genomic DNA, which protocol not only provides ccc DNA of a high purity grade but also removes proteinaceous impurities. More specifically, the suggested method comprises to precipitate a cleared lysate with alcohol; to wash the precipitate with an alcohol solution; to resuspend the precipitate; to digest the resuspended precipitate with a RecBCD nuclease (EC 3.1.11.5); and to separate purified ccc DNA from the remainder of the product obtained by contacting it with an ion exchange material. RNAse may be added to the cleared lysate, and the precipitation step is stated to separate the DNA of the cells from other components, including RNA and proteins.
U.S. Pat. No. 6,498,236 (Upfront Chromatography A/S) relates to a method for the isolation of immunoglobulins from a solution, which method allows high efficiency and use of little or no salts, especially lyotropic salts. Solid phase matrices are used, preferably epichlorohydrin activated agarose matrices, which have been functionalised with aromatic or heteroaromatic ligands which preferably comprises an acidic substituent such as a carboxylic acid, i.e. a weak cation exchange group. Alternatively, the matrix backbones are dextran-based, such as Sephadex™, cellulose-based, such as Perloza™ cellulose, composite beads, such as Sephacryl™ and Superdex™, synthetic origin beads such as Fractogel™ etc.
Further, U.S. Pat. No. 6,572,766 (Amersham Biosciences) discloses a separation matrix comprising a core showing a micropore system and a surface in which the pore system has openings, wherein the surface is covered with a polymer which exhibits such a large molecular weight that it cannot penetrate into the micropore system. Thus, the polymer has a molecular weight distribution of such a kind that all or substantially all polymer molecules in the preparation are excluded from transport into the micropores, when the preparation is contacted with a liquid which can be transported into the matrix. The polymer may be functionalised with a different ligand from the micropore surfaces. This means that the polymer, when it is anchored to the outer surface, can give separation characteristics to the surface, which are different from the separation characteristics of the micropores. The method is suggested for separation of nucleic acid, proteins including peptides and other organic and inorganic compounds.
Finally, WO 01/37987 (Amersham Biosciences) relates to the separation of negatively charged nucleic acids from each other and from other negatively charged components such as proteins. More specifically, disclosed is a the use of an adsorbent that exhibits an interior part, which carries a ligand structure capable of binding to substances I and II, and is accessible to substance I, and an outer surface layer which is free from ligand structures and which is more easily penetrated by substance I than by substance II. Thus, since there are no ligands present on the outer surface layer, i.e. the external surface, adsorption will be limited to the interior part, i.e. the pore surfaces. The outer surface may even carry repelling structures. Thus, substance I will be adsorbed within the adsorbent, while structure II will pass through the column without being adsorbed. Substance I and/or II may exhibit a nucleic acid structure. The process is e.g. useful for separating linear DNA from circular DNA, RNA from plasmids, plasmids from genomic DNA, plasmids from endotoxins etc. The process is optionally followed by further steps. For example, if substance II is desired in a highly purified form, it should be followed by an additional capture step, such as ion exchange, revered phase chromatography (RPC), HIC etc.
However, there is still a need in this field of alternative purification schemes enabling isolation of large target compounds, such as plasmids, at high productivity and selectivity.